The neural crest is a multipotent embryonic cell type that arises in the dorsal neural tube and migrates into the periphery to form a variety of structures including most of the craniofacial skeleton. Efforts to characterize neural crest formation at the molecular level have focused on the transcription factors that specify neural crest cells in the ectoderm. However, neural crest gene expression does not guarantee eventual migration as a neural crest cell. We propose that differential protein activity, rather than differential gene expression, regulates neural crest migratory properties. One possibility is that the phosphorylation status of proteins important for neural crest migration determines the ability of neural crest cells to migrate. We have identified a putative phosphatase, paladin, which is expressed by premigratory neural crest cells and upregulated at the initiation of neural crest migration. Knockdown of this putative phosphatase in chick embryos does not affect the timing of neural crest specification, but delays expression of snall-2, a transcription factor important for neural crest migration, and inhibits neural crest migration. From these data / have formed the hypothesis that Paid Is a phosphatase that regulates proteins that control neural crest EMT and migration. In order to more fully characterize the role of paladin in neural crest development, we have generated mice mutant for paladin. In Specific Aim 1, I will characterize defects in neural crest development and migration in paladin mutant mice. In Specific Aim 2, I will use function and activity-based assays to determine if paladin is a phosphatase or a phosphoprotein-binding antiphosphatase. Finally, in Specific Aim 3, I will conduct a modified yeast-two hybrid to identify targets and binding partners of paladin. These studies will allow us to better understand how migratory neural crest cells are generated and therefore better understand the early steps ih craniofacial development, giving us more strategies for prevention and treatment of neural crest-derived birth defects. Public Health Relevance: Neural crest cells are an embryonic stem cell-like population that forms a variety of different structures in the adult animal, including the bones of the skull. Defects in neural crest development cause birth defects in 1.3% of live human births. Our goal is to understand how neural crest cells form, which will help us to prevent and treat neural crest-derived birth defects.